Lymphocyte T Regulatory , Th17 and NKT in Epithelial Ovarian Tumor- Prognostic Assessment and Relationship With Clinical Marker

Study groups. The study group consisted of 60 women. The patients were divided into 3 subgroups: a group of 24 women with malignant epithelial ovarian tumors (cystadenocarcinomas), 25 women with benign ovarian tumors (cystadenomas) and 11 women with borderline ovarian tumors (serous borderline tumors). The control group consisted of 20 women without ovarian pathology who underwent surgery for unexplanied infertility. Patients were admitted to IInd Department of Gynecology, Lublin Medical University, Lublin, Poland, between 2011 and 2014. All women with OCs were staged III or IV according to revised 2013 FIGO classification (International Journal of Gynecology and Obstetrics, January, 2014) Isolation of mononuclear cells from peripheral blood (PBMCs). Immediately after being taken from the antecubital vein, mononuclear cells (PBMCs) were isolated by density gradient centrifugation applying Gradisol L formulation of specific gravity 1.077g/ml (Aqua Medica, Łódź, Poland) for 20 min. at 700 x g. The pellet containing PBMC was washed twice in PBS and evaluated for the number (using Neubauer chamber) and viability (trypan blue staining - 0.4% trypan Blue Solution, Sigma-Aldrich, Munich, Germany). Viability of less than 95% was a disqualifying criterion.

Isolation of mononuclear cells infiltrating the tumor and healthy tissues.

During the surgery, ovarian tissue (size of approximately 1cm3) not containing any visible necrotic areas was collected. It was minced with a scalpel, suspended in 30 ml of RPMI 1640 medium (Biochrome, Holliston MA, USA) and subjected to cleavage in a mixture containing: 1 mg/ml collagenase type IA (Sigma-Aldrich, Munich, Germany), 1 mg/ml DNase type I (Sigma-Aldrich, Munich, Germany), 0.1 mg/ml hyaluronidase (Sigma-Aldrich, Munich, Germany) at 37°C for 60 min., constantly vortexed. After cleavage, the suspension was filtered through a strainer (70μm, BD Biosciences, San Jose CA, USA) and centrifuged for 5 min. at 700 x g. The suspension cells were washed twice in RPMI 1640.

Evaluation of the percentage of Th17 cells.

The evaluation of the percentage of Th17 cells (secreting IL-17A) of peripheral blood mononuclear cells, healthy tissue and tumor tissue was performed by flow cytometry using a Th17 Cytokine Staining panel according to the manufacturer's recommendation (eBiosciences, San Diego, USA) using a FACSCanto (BD Biosciences, San Jose CA,USA).